The long-term aim of this work is to understand the structural components of the BK-type calcium (Ca2+)-activated potassium (K+) channel complex, the functional properties of the channels, and the physiological roles of those channels. BK channels are widely expressed among different cell types and exhibit significant functional diversity suited to their physiological roles. BK currents couple changes in submembrane Ca2+ concentrations to changes in membrane potential and excitability. They consist minimally of four pore-forming subunits. In addition, there are four auxiliary subunits, which differ not only in terms of tissue distribution, but also in terms of the functional properties of the resulting BK channels. Two auxiliary subunits, the 2 and 3b, produce a novel rapid inactivation of BK channel current. This project will focus specifically on the mechanism and structural basis of inactivation mediated by these subunits. Inactivation resulting from these subunits involves a two-step mechanism unlike any inactivation mechanism so far described. Using methods of electrophysiology combined with molecular biology, this project will determine the structural components and molecular steps involved in the inactivation mechanism produced by the 2 and 3b subunits. First, a model by which inactivation domains bind to a pore constriction at the mouth of the channel will be functionally tested. Second, the points of interaction between inactivation domains and their binding sites will be determined. Third, the validity of two-step inactivation mechanism will be confirmed and the molecular nature of each of the two steps in the inactivation process determined. These studies will provide important information about key structural changes associated with BK channel gating and the structural components that contribute to the channel near the channel pore. BK channels are of broad importance in the normal functioning of a variety of excitable cells. Among different tissues, BK channels contribute to regulation of neuronal excitability, smooth muscle relaxation, synaptic transmission and hormone release. Better understanding the composition and functional role of BK channel variants is of potential medical importance, not only because the channels may serve as specific therapeutic targets but also because altered function of particular variants may contribute to unknown pathological conditions.